蛋白C基因新突变的分子发病机制研究

目的对一个新发现的蛋白C（protein C,PC）基因突变PC E29K进行体外表达研究以探讨其导致PC缺陷的分子发病机制。方法采用定点突变方法构建PC E29K突变表达质粒,脂质体法转染COS-7细胞、培养。采用ELISA法测定培养上清液和细胞裂解液中的PC：Ag,用激光共聚焦显微镜对培养细胞进行细胞免疫荧光染色分析。结果转染了突变质粒的Cos-7细胞培养上清中PC：Ag的含量为野生型的23.7%,细胞裂解液中PC：Ag的含量为野生型的81.1%。PC E29K突变型蛋白多在内质网中滞留,在高尔基体中定位减少。结论 PC E29K仅有部分从细胞内分泌出来,且部分在细胞内降解,因此,分泌障碍和部分降解加速是该突变导致PC缺陷的直接原因。

Study on the Molecular Pathogenesis Underlying a Novel Protein C Mutation

Objective To study the molecular pathogenesis underlying a novel protein C(PC) E29K gene mutation through in vitro expression analysis. Methods Expression vector of PC E29K mutation was constructed by site-directed mutation method using wild-type PC plasmid as template.The plasmids were transfected into COS-7 cells by lipofectin transfection method and cultured.PC antigen levels of the supernatant and cell lysis medium were measured by ELISA method.Laser confocal microscope was used to detect the cellular immunofluorecent localization of the cultured cells. Results PC antigen level of the supernatant of COS-7 transfected with PC E29K plasmid was 23.7% of that of wild-type ones.The PC antigen level in the cell lysis medium of COS-7 cells transfected with mutant plasmid was 81.1% of that of the wild-type.PC E29K mutated protein was mostly trapped in the endoplasmic reticulum and decreased in Golgi bodies. Conclusion As only part of PC E29K mutated protein was secreted from the cells and part of it was degraded inside the cells,impaired secretion and partial degradation is the cause of this mutation.